影响小鼠孤雌胚胎干细胞的建系效率因素初探

目的检测孤雌胚胎干细胞系的建系效率与小鼠品系以及培养体系的关系。方法将小鼠MⅡ期卵子孤雌激活发育至囊胚,然后从囊胚内细胞团分离孤雌胚胎干细胞。结果杂交和近交系小鼠的建系效率没有显著差异,建系的培养体系中加入ERK抑制剂或者采用血清替代品KSR时,建系效率显著提高。结论小鼠孤雌胚胎干细胞的建系效率与小鼠的遗传背景并没有直接关系,而与分离内细胞团的培养体系密切相关。     

印迹基因修饰使孤雌胚胎干细胞获得四倍体补偿能力

孤雌胚胎干细胞(Parthenogenetic embryonic stem cells,pESCs)的遗传物质全部来源于母源基因组,因缺失父源基因而不具备四倍体补偿的能力。为了使pESCs也具备发育到个体的能力,呈现与受精卵来源ESCs类似的多能性,文中借助CRISPR/Cas9系统对孤雌来源的pESCs中的2个重要母源印迹基因的差异甲基化区域(Differentially methylated region,DMR)进行单等位基因敲除(H19-DMR,IG-DMR),获得双基因敲除的(DKO)pESCs。结果表明,pESCs虽然来源于母源基因组,但是其形态特征、多能干性标记分子的表达水平、体外神经分化能力与受精卵来源的ESCs基本一致。最后,通过基因修饰的DKOpESCs可以通过四倍体补偿获得发育到期的胎儿,表明经过印迹基因修饰的pESCs也具有发育到一个完整个体的多能性。从而为再生医学研究提供了一类具有主要组织相容性复合基因匹配且多能性良好的资源细胞。     

小鼠孤雌胚胎干细胞系的建立及生物学特性鉴定

目的:探讨建立合适的小鼠孤雌胚胎干细胞建系方法。方法:采用氯化锶联合细胞松弛素B激活B6D2F1杂交小鼠卵母细胞,所获得的囊胚与桑椹胚分别用于孤雌胚胎干细胞的建系,观察两者的建系成功率。结果:共建立了12株小鼠孤雌胚胎干细胞系,这些细胞SSEA-1抗原阳性,SSEA-4,TRA-1-81,TRA-1-60表面抗原阴性,具有AKP活性,保持正常染色体核型,体内外分化分别形成畸胎瘤和拟胚体。结论:采用囊胚和去透明带的桑葚胚建立孤雌胚胎干细胞系获得成功。该方法为人类纯合子的胚胎干细胞建系提供基础,在自体细胞治疗领域中具有潜在的应用价值。     

小鼠孤雌胚胎干细胞集落的建立

ＥＳＴＡＢＬＩＳＨＭＥＮＴＯＦＳＴＥＭＣＥＬＬＣＯＬＯＮＩＥＳＦＲＯＭＰＡＲＴＨＥＮＯＧＥＮＥＴＩＣＡＬＬＹＤＥＲＩＶＥＤＢＬＡＳＴＯＣＹＳＴＳＯＦＭＯＵＳＥ小鼠孤雌胚胎干细胞集落的建立ＫｅｙｗｏｒｄｓＭｏｕｓｅ,Ｐａｒｔｈｅｎｏｇｅｎｅｔｉｃｅｍ...     

哺乳动物孤雌干细胞与孤雌生殖

未受精的孤雌胚胎衍生的孤雌胚胎干细胞(parthenogenetic embryonic stem cells,pESCs),具有与胚胎干细胞(embryonic stem cells,ESCs)相似的多向分化和自我更新能力,且具备来.源广泛、获取高效及低致瘤性等优势,因此成为近年来的研究热点.该文概述了pESCs特殊...     

胚胎干细胞向肝细胞诱导分化

胚胎干细胞具有分化成三胚层细胞的潜能。它已被视为治疗多种疾痛的一种新兴策略。在现阶段,通过不同的诱导途径可将胚胎干细胞诱导成为肝细胞：体外诱导、体内诱导以及体外和体内相结合诱导分化。然而从体内实验结果来看,其嵌合率及分化率不高,这是一个亟需解决的问题,否则就无法成功地将其应用于临床治疗。     

胚胎干细胞向血液干细胞定向分化的研究进展

骨髓移植是目前治疗恶性白血病以及遗传性血液病最有效的方法之一。但是HLA相匹配的骨髓捐献者严重短缺,骨髓造血干细胞（hematopoietic stem cells,HSCs）体外培养困难,在体外修复患者骨髓造血干细胞技术不成熟,这些都大大限制了骨髓移植在临床上的应用。多能性胚胎干细胞（embryonic stem cells,ESCs）具有自我更新能力,在合适的培养条件下分化形成各种血系细胞,是造血干细胞的另一来源。在过去的二十多年里,血发生的研究是干细胞生物学中最为活跃的领域之一。小鼠及人的胚胎干细胞方面的研究最近取得了重大进展。这篇综述总结了近年来从胚胎干细胞获得造血干细胞的成就,以及在安全和技术上的障碍。胚胎干细胞诱导生成可移植性血干细胞的研究能够使我们更好地了解正常和异常造血发生的机制,同时也为造血干细胞的临床应用提供理论和实验依据。     

小鼠胚胎干细胞向生殖细胞分化的研究

小鼠胚胎干细胞(ESC)在体外可以分化为多种细胞类型,其中包括各阶段的生殖细胞,甚至精细胞和成熟卵母细胞。ESC向生殖细胞分化的效率受到包括生长因子、激素和体细胞等多种因素的影响,在体外形成的是雌性配子还是雄性配子与ESC是XX型还是XY型没有必然联系。简要综述了小鼠生殖细胞在体内外的分化发育、性别决定和增殖等,并总结和展望了ESC向生殖细胞分化研究面临的问题和应用前景。     

人孤雌胚胎干细胞在人包皮成纤维细胞饲养层上的生长状态

人孤雌胚胎干细胞(human parthenogenetic embryonic stem cells,hPESCs)体外培养常需饲养层的支持以保持干细胞特性.通过原代培养获得人包皮成纤维细胞(human foreskin fibroblasts,hFFs)并将其制备成饲养层,使hPESCs在hFFs上进行体外培养及传代.倒置显微镜下观察hPESCs的生长状态,采用碱性磷酸酶(alkalinephosphatase,AKP)检测、核型分析和体内分化实验研究hPESCs的生物学特性及分化潜能,以探索hFFs能否长期支持hPESCs的生长并维持其未分化状态.经原代培养成功获得了hFFs,通过形态学观察和免疫细胞化学染色鉴定符合成纤维细胞的生物学特性;在hFFs上生长的hPESCs克隆形态规则,不易分化;已成功在体外培养20余代,hPESCs仍能够保持基本生物学特性和正常核型,在裸鼠体内可形成含有3个胚层组织成分的畸胎瘤.作为人源性饲养层,hFFs可长期支持hPESCs的生长并维持其未分化状态.     

成熟软骨细胞促进小鼠胚胎干细胞向软骨分化

为诱导胚胎干细胞向软骨细胞分化,采用胚胎干细胞与成熟软骨细胞共培养的方法以提供软骨诱导微环境．小鼠胚胎干细胞经初步分化,形成类胚体后,从中分选出Flk-1阳性细胞,其与猪关节软骨细胞混合后,接种于可降解材料支架,并植入裸鼠皮下．4周后经组织学检查及Ⅱ型胶原抗体免疫荧光检查证实,细胞材料复合物形成软骨组织．经小鼠主要组织相容性抗原染色,证实其中部分软骨细胞源自于小鼠胚胎干细胞．本研究建立了胚胎干细胞定向诱导分化的新方法．     

Efficient differentiation of mouse embryonic stem cells into motor neurons

Direct differentiation of embryonic stem (ES) cells into functional motor neurons represents a promising resource to study disease mechanisms, to screen new drug compounds, and to develop new therapies for motor neuron diseases such as spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). Many current protocols use a combination of retinoic acid (RA) and sonic hedgehog (Shh) to differentiate mouse embryonic stem (mES) cells into motor neurons. However, the differentiation efficiency of mES cells into motor neurons has only met with moderate success. We have developed a two-step differentiation protocol that significantly improves the differentiation efficiency compared with currently established protocols. The first step is to enhance the neuralization process by adding Noggin and fibroblast growth factors (FGFs). Noggin is a bone morphogenetic protein (BMP) antagonist and is implicated in neural induction according to the default model of neurogenesis and results in the formation of anterior neural patterning. FGF signaling acts synergistically with Noggin in inducing neural tissue formation by promoting a posterior neural identity. In this step, mES cells were primed with Noggin, bFGF, and FGF-8 for two days to promote differentiation towards neural lineages. The second step is to induce motor neuron specification. Noggin/FGFs exposed mES cells were incubated with RA and a Shh agonist, Smoothened agonist (SAG), for another 5 days to facilitate motor neuron generation. To monitor the differentiation of mESs into motor neurons, we used an ES cell line derived from a transgenic mouse expressing eGFP under the control of the motor neuron specific promoter Hb9. Using this robust protocol, we achieved 51 ± 0.8% of differentiation efficiency (n = 3; p < 0.01, Student's t-test). Results from immunofluorescent staining showed that GFP+ cells express the motor neuron specific markers, Islet-1 and choline acetyltransferase (ChAT). Our two-step differentiation protocol provides an efficient way to differentiate mES cells into spinal motor neurons.     

Progress in studies on the induction and differentiation of embryonic stem cells

Embryonic stem cells (ESCs), which are isolated from the inner cell mass of the blastocyst stage embryo, have the potential to give rise to an entire organism and to generate every body cell type. Much improvement has been made in the field of induction and differentiation of ESCs during the last two years, such as the ESCs differentiation into germ cells (2003) and the cloning of human ESCs (2004), both of which were chosen respectively as one of the top ten achievements evaluated by academic journals. Great attention was also paid to the research of the new genes which could maintain ESCs in the undifferentiated state and the research of the induction and differentiation of ESCs.     

A simplified method to generate serotonergic neurons from mouse embryonic stem and induced pluripotent stem cells

We have developed a new simple method to induce serotonergic neurons from embryonic stem (ES) and induced pluripotent stem cells. When ES or induced pluripotent stem cells were cultured on a thick gel layer of Matrigel, most colonies extended TuJ1-positive neurites. We found that noggin, a known antagonist of bone morphogenic protein, induces ES cells to express genes involved in serotonergic differentiation, such as Nkx2.2, Pet-1, Sonic hedgehog, tryptophan hydroxylase 2, and serotonin transporter, as well as increases high potassium-induced release of serotonin. To concentrate serotonergic neurons, ES cells carrying Pet-1-enhancer-driven enhanced green fluorescent protein were differentiated and sorted into about 80% pure cultures of serotonergic neurons. Whole cell voltage-clamp recordings showed a voltage-dependent current in dissociated neurons. This simplified method provides an alternative option for serotonergic differentiation of pluripotent stem cells and will likely contribute a deeper understanding regarding the nature of serotonergic neurons and open new therapeutic perspectives for the treatment of psychiatric disorders.     

Retinoic-acid-concentration-dependent acquisition of neural cell identity during in vitro differentiation of mouse embryonic stem cells

Retinoic acid (RA) is one of the most important morphogens, and its embryonic distribution correlates with neural differentiation and positional specification in the developing central nervous system. To investigate the concentration-dependent effects of RA on neural differentiation of mouse embryonic stem cells (ES cells), we investigated the precise expression profiles of neural and regional specific genes by ES cells aggregated into embryoid bodies (EBs) exposed to various concentrations of RA or the BMP antagonist Noggin. RA promoted both neural differentiation and caudalization in a concentration-dependent manner, and the concentration of RA was found to regulate dorso-ventral identity, i.e., higher concentrations of RA induced a dorsal phenotype, and lower concentrations of RA induced a more ventral phenotype. The induction of the more ventral phenotype was due to the higher expression level of the N-terminus of sonic hedgehog protein (Shh-N) when treated with low concentration RA, as it was abrogated by an inhibitor of Shh signaling, cyclopamine. These findings suggest that the concentration of RA strictly and simultaneously regulates the neuralization and positional specification during differentiation of mouse ES cells and that it may be possible to use it to establish a strategy for controlling the identity of ES-cell-derived neural cells.     

Progress in studies on the induction and differentiation of embryonic stem cells

Embryonic stem cells (ESCs), which are isolated from the inner cell mass of the blastocysts, have the potential to give rise to an entire organism and to gen-erate every body cell type. During the last two years, much progress has been made in ESCs field, espe-cially in the induction and differentiation of ESCs. 1 ESCs differentiate into cells of different types 1.1 ESCs differentiate into germ cells In 2003, it was reported that mouse ESCs could differentiate into oocyte[1]. Oct-4 was …     

Aggregation of pre-implantation embryos improves establishment of parthenogenetic stem cells and expression of imprinted genes

Parthenogenetic embryonic stem cells (PgES) might advance cell replacement therapies and provide a valuable in vitro model system to study the genomic imprinting. However, the differential potential of PgES cells was limited. It could result from relative low heterology of PgES cells compared with ES cells from fertilization (fES), which produce different expression of most imprinted genes. Here, we described the establishment of PgES cells by aggregating parthenogenetic embryos at the 8-cell stage (aPgES cells), which may increase heterozygy. We found that derivation of aPgES cells in association with an increased number of inner cell mass cells by aggregating was more efficient than that of PgES cells from a single parthenogenetic blastocyst. The aPgES cells have normal karyotype, stain positive for alkaline phosphatase, express high levels of ES cell markers and can differentiate into teratomas composed of the three germ layers. Moreover, compared with PgES cells, the more highly upregulated paternally expressed imprinted genes were observed in aPgES cells, the same change was not shown in aPg blastocysts. This suggested that the aggregation induced effect could modify the expression of paternally expressed imprinted genes. Our studies showed that aPgES cells, the expression of imprinted genes in which more closely resemble fES cells than PgES cells, would contribute to all organs and avoiding immuno-rejection, which may provide invaluable material for regeneration medicine.